Recombinant Chicken Calcium release-activated calcium channel protein 1 (ORAI1)
Description
Structure and Function
ORAI1 channels are activated by the "store-operated" or "capacitative" mechanism following the depletion of internal calcium stores . When phospholipase C is activated by cell surface receptors, inositol trisphosphate is produced, which then triggers the release of calcium from the ER . The STIM1 protein detects the decreased calcium concentration in the ER . Upon calcium store depletion, STIM1 clusters and forms puncta, relocating near the plasma membrane, where it activates ORAI1 through protein-protein interaction .
ORAI1 is the pore-forming subunit of the CRAC channel, essential for Ca2+ signaling in mammalian cells . Electrophysiological data has revealed that acidic residues like E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to ORAI1 channels' high selectivity for Ca2+ .
A 3.35-angstrom (Å) crystal structure of the Drosophila Orai channel, which shares 73% sequence identity with human Orai1 within its transmembrane region, was published in 2012 . The structure, representing the channel's closed state, showed that a single channel comprises six Orai subunits, with the transmembrane domains arranged in concentric rings around a central aqueous pore formed by the first transmembrane helix of each subunit . Transmembrane helices 2 and 3 surround TM1, shielding it from the lipid bilayer and providing structural support, while the fourth transmembrane helix forms the outermost layer .
Mechanism
Under resting conditions, Orai1 is located at the plasma membrane (PM), while STIM1 resides in the ER membrane . Ca2+-bound STIM1 adopts a folded structure mediated by an intramolecular interaction . Upon store depletion, STIM1 unfolds, oligomerizes, and translocates to form clusters at junctional regions .
Orai1 redistribution into the ER–PM junctions depends on interaction with STIM1 . STIM1 redistribution into the junctions is more complex and modulated by protein interactors like ERp57, P100, golli, and CRACR2A . The polybasic residues in the C terminus of STIM1 are crucial for STIM1 clustering at the ER–PM junctions through interaction with PM phosphoinositides . Positive regulators of SOCE (e.g., CRACR2A) also form a complex with Orai1 and STIM1 to stabilize their interaction . After intracellular Ca2+ concentration ([Ca2+]i) increases, negative regulators of SOCE, such as calmodulin, interact with Orai1 to inactivate the CRAC channels . When ER Ca2+ is refilled by SERCA, the protein complex of Orai1 and STIM1 dissociates .
Interacting Partners
Numerous molecules are involved in regulating CRAC channel activity under physiological conditions . Genome-wide RNAi screens for CRAC channel components have yielded hundreds of positive hits . Orai1 exists in a macromolecular complex with an 11–14 nm protrusion into the cytoplasm . Biochemical analyses have identified Orai1 and STIM1 in a macromolecular protein complex . Numerous interacting partners of Orai1 and STIM1 regulate various stages of CRAC channel activation .
Role in Disease
Loss-of-function mutations in Orai1 can cause severe combined immunodeficiency (SCID) . Orai1 plays a role in the activation of T-lymphocytes, and defects in this gene can cause immune dysfunction with T-cell inactivation due to calcium entry defect type 1 (IDTICED1) .
Orai1 and Bone Development
Orai1 plays a central role in regulating bone cells . Studies with mice have demonstrated that the absence of Orai1 inhibits osteoclast and osteoblast differentiation, impairing skeletal development . Orai1 knockout mice lacked multinucleated osteoclasts but did not develop osteopetrosis . These mice showed decreased bone formation with retention of fetal cartilage, reduced cortical ossification, and thinned trabeculae . Orai1 is essential for the differentiation and function of human osteoblasts, highlighting its critical role in bone development .
Orai1 and Cell Migration
Calcium entry through freely diffusing TRPV1 channels induces strong calcium-dependent inactivation (CDI) on Orai1, while calcium entering through P2X4 channels does not . TRPV1 and Orai1 channels are in close proximity in the cell membrane, allowing TRPV1 to induce CDI on Orai1 . TRPV1-mediated CDI on Orai1 plays a role in cell migration and wound healing .
Product Specs
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Target Background
Q&A
Basic Research Questions
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What is Chicken ORAI1 and what is its function in calcium signaling?
ORAI1 (also known as CRACM1, Calcium release-activated calcium channel protein 1, and TMEM142A) is a calcium selective ion channel encoded by the ORAI1 gene. It plays a critical role in store-operated calcium entry (SOCE), which is essential for calcium influx into cells following depletion of intracellular calcium stores. In chickens, as in mammals, ORAI1 forms a channel in the plasma membrane that mediates calcium influx, particularly important for T-lymphocyte activation. Loss of function mutations in ORAI1 can cause severe combined immunodeficiency (SCID) in humans, highlighting its evolutionary conservation and importance .
The protein belongs to a family that includes two additional homologs, ORAI2 and ORAI3. ORAI proteins share no homology with other ion channel families or known proteins. Structurally, they have 4 transmembrane domains and form tetramers, with conserved acidic residues in the transmembrane segments that are critical for calcium selectivity .
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How is recombinant chicken ORAI1 protein typically produced for research purposes?
Recombinant chicken ORAI1 protein is typically produced using a baculovirus expression system, which enables proper folding and post-translational modifications essential for membrane proteins. The process involves:
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Cloning the chicken ORAI1 gene sequence (partial or complete) into a baculovirus expression vector
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Transfecting insect cells with the recombinant vector
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Viral amplification and protein expression in the insect cell system
This expression system is preferred for membrane proteins like ORAI1 that require eukaryotic cellular machinery for proper folding and potential post-translational modifications. The resulting recombinant protein (e.g., product code CSB-BP720018CH1) can then be used for various research applications, including antibody generation, protein-protein interaction studies, and functional assays .
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What are the optimal storage and handling conditions for recombinant chicken ORAI1?
Maintaining the structural integrity and functional properties of recombinant chicken ORAI1 requires specific storage and handling protocols:
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Storage temperature: -20°C to -80°C is recommended for both liquid and lyophilized forms
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Shelf life: Approximately 6 months for liquid formulations and up to 12 months for lyophilized forms
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Reconstitution procedure:
Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as it can lead to protein denaturation and activity loss . These precautions help preserve the native conformation and functional properties of the protein for experimental applications.
Advanced Research Questions
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How does T cell-specific deletion of ORAI1 affect immune responses in different disease models?
Research using conditional knockout models (Orai1fl/flCd4Cre mice) has revealed fascinating differential roles of ORAI1 in various immune contexts:
These findings demonstrate that ORAI1 plays differential roles in T cell-mediated immunity to infection versus inflammatory responses. This suggests potential therapeutic approaches targeting ORAI1 in allergic diseases without compromising anti-viral immunity .
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What experimental approaches are most suitable for investigating the role of chicken ORAI1 in T cell activation?
Investigating chicken ORAI1's role in T cell activation requires a multi-faceted experimental approach:
| Technique | Methodology | Information Gained |
|---|---|---|
| Calcium imaging | Use of fluorescent calcium indicators (e.g., Fura-2) to monitor real-time changes in intracellular calcium | Direct visualization of SOCE in response to store depletion or receptor stimulation |
| Patch-clamp electrophysiology | Whole-cell recordings in primary chicken T cells or heterologous expression systems | Precise measurements of CRAC channel currents, kinetics, and ion selectivity |
| Genetic manipulation | CRISPR/Cas9 editing or RNA interference to modify ORAI1 expression | Effect of ORAI1 deletion or mutation on T cell function |
| Protein-protein interaction assays | Co-immunoprecipitation, FRET, or proximity ligation assays | Interaction between chicken ORAI1 and regulatory proteins like STIM1 |
| T cell functional assays | Measurement of proliferation, cytokine production, and differentiation | Downstream consequences of ORAI1 activity in T cell immunity |
These complementary approaches provide a comprehensive understanding of ORAI1's role in chicken T cell calcium signaling and immune function, allowing researchers to draw parallels with mammalian systems while identifying avian-specific mechanisms .
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How do mutations in conserved acidic residues affect chicken ORAI1 channel function?
Studies on ORAI1 have identified critical acidic residues in the transmembrane domains that determine calcium selectivity and channel function. In human ORAI1, mutations E106D in transmembrane helix 1 and E190Q in transmembrane helix 3 significantly alter channel properties .
Based on the high conservation of these functional domains, similar mutations in chicken ORAI1 would be expected to:
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Diminish calcium ion influx
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Increase current carried by monovalent cations
These mutations provide powerful tools for structure-function studies of chicken ORAI1, allowing researchers to:
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Map the ion conduction pathway
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Identify species-specific differences in channel regulation
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Study the molecular basis of calcium selectivity
Experimental approaches to study these mutations include site-directed mutagenesis followed by functional characterization in heterologous expression systems or chicken T cells using calcium imaging and electrophysiology .
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What are the experimental considerations when comparing T cell-mediated immunity in viral infections versus allergic inflammation models?
The differential role of ORAI1 in antiviral immunity versus allergic inflammation raises important experimental considerations:
When investigating ORAI1 function across these models, researchers should consider using:
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OVA-specific T cell receptor transgenic models to control for antigen specificity
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T cell transfer experiments to isolated T cell-intrinsic effects
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Recombinant cytokine administration (e.g., IL-2) to rescue specific signaling defects
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What methods can be used to study the interaction between recombinant chicken ORAI1 and its regulatory proteins?
Studying interactions between chicken ORAI1 and its regulatory proteins (particularly STIM1) requires specialized techniques for membrane protein analysis:
| Method | Technical Approach | Advantages | Limitations |
|---|---|---|---|
| Co-immunoprecipitation | Pull-down of protein complexes using specific antibodies | Detects native protein interactions | May disrupt weak interactions during cell lysis |
| Förster Resonance Energy Transfer (FRET) | Measurement of energy transfer between fluorophore-tagged proteins | Real-time monitoring in live cells | Requires protein tagging which may affect function |
| Proximity Ligation Assay | Antibody-based detection of proteins in close proximity | High sensitivity, works with endogenous proteins | Requires highly specific antibodies |
| Surface Plasmon Resonance | Detection of binding between purified proteins | Provides binding kinetics and affinity | Requires purified proteins in native conformation |
| Bioluminescence Resonance Energy Transfer (BRET) | Energy transfer between luciferase and fluorescent protein tags | Good for membrane protein interactions | Requires protein tagging |
These methods can reveal:
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The stoichiometry of chicken ORAI1-STIM1 complexes
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Conformational changes during channel activation
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Species-specific differences in regulatory interactions
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Effects of mutations on protein-protein binding
For recombinant chicken ORAI1, ensuring proper protein folding and membrane insertion is critical for meaningful interaction studies .
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How can recombinant chicken ORAI1 be used to develop specific inhibitors for research applications?
Recombinant chicken ORAI1 provides a valuable tool for developing and screening specific inhibitors through several approaches:
| Screening Approach | Methodology | Advantages |
|---|---|---|
| High-throughput calcium flux assays | Measure SOCE inhibition in cells expressing recombinant chicken ORAI1 | Rapid screening of large compound libraries |
| Structure-based virtual screening | In silico docking of compounds to predicted ORAI1 structure | Cost-effective initial screening |
| Electrophysiological validation | Patch-clamp confirmation of promising compounds | Direct functional assessment of channel block |
| Species-comparative screening | Testing compounds against chicken vs. mammalian ORAI1 | Identification of species-selective inhibitors |
The development process should include:
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Primary screening using calcium imaging in heterologous expression systems
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Secondary validation with electrophysiology
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Specificity testing against other calcium channels
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Assessment in primary chicken T cells
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In vivo validation in appropriate avian models
Such inhibitors would serve as valuable tools for studying ORAI1 function in avian immune responses and calcium signaling pathways .
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What bioinformatic approaches can predict functional domains and regulatory elements in chicken ORAI1?
Comprehensive bioinformatic analysis of chicken ORAI1 can provide valuable insights into its structure and function:
| Bioinformatic Approach | Tools | Application to Chicken ORAI1 |
|---|---|---|
| Sequence alignment | BLAST, Clustal Omega, MUSCLE | Identify conserved domains across species |
| Phylogenetic analysis | MEGA, PhyML, MrBayes | Evolutionary relationships between ORAI homologs |
| Transmembrane topology prediction | TMHMM, Phobius, TOPCONS | Define membrane-spanning regions |
| Protein domain prediction | SMART, Pfam, InterPro | Identify functional domains and motifs |
| Post-translational modification sites | NetPhos, NetOGlyc, GPS | Predict regulatory modification sites |
| 3D structure prediction | AlphaFold, I-TASSER, SWISS-MODEL | Model tertiary structure of chicken ORAI1 |
| Protein-protein interaction sites | SPPIDER, PredictProtein | Identify potential STIM1 binding regions |
These computational approaches can:
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Guide the design of site-directed mutagenesis experiments
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Identify chicken-specific structural features
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Predict functional consequences of naturally occurring variants
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Inform the design of chicken-specific antibodies or inhibitors
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What are the technical challenges in measuring CRAC channel currents in chicken T cells?
Electrophysiological characterization of native CRAC channels in chicken T cells presents several technical challenges:
| Challenge | Technical Consideration | Potential Solution |
|---|---|---|
| Small cell size | Difficult to establish stable patch-clamp configurations | Use of optimized pipette geometry and recording solutions |
| Low current amplitude | Native CRAC currents are typically only a few pA | Background noise reduction and signal averaging techniques |
| Channel rundown | Loss of channel activity during prolonged recordings | Optimized internal solutions with appropriate calcium buffers |
| Cell heterogeneity | Variable channel expression in primary T cell populations | Cell sorting for specific T cell subsets before recording |
| Distinguishing from other calcium currents | Multiple calcium channels may be present | Pharmacological isolation with specific blockers |
Researchers can overcome these challenges by:
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Using recombinant systems for initial characterization
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Employing perforated-patch techniques to preserve intracellular signaling
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Combining electrophysiology with fluorescence imaging
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Using specific CRAC channel activators like STIM1 fragments
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Comparing results with mammalian systems where CRAC currents are well-characterized
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How does ORAI1 function differ between chicken T cell subsets, and what methods can investigate these differences?
Investigating ORAI1 function across different chicken T cell subsets requires specialized techniques:
| T Cell Subset | Expected ORAI1-Dependent Functions | Investigative Methods |
|---|---|---|
| CD4+ helper T cells | Cytokine production, proliferation, differentiation | - Subset-specific isolation followed by calcium imaging - Cytokine profiling after TCR stimulation - In vitro differentiation assays |
| CD8+ cytotoxic T cells | Granule release, target cell killing, memory formation | - Cytotoxicity assays with calcium chelation - Degranulation measurement (CD107a exposure) - Intracellular cytokine staining |
| Memory T cells | Rapid recall responses, sustained calcium signaling | - Memory cell isolation from previously immunized birds - Calcium response kinetics in memory vs. naive cells |
| γδ T cells | Innate-like responses, tissue surveillance | - Comparative calcium signaling in γδ vs. αβ T cells - Analysis of tissue-resident subsets |
Research approaches should include:
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Flow cytometry-based calcium flux analysis with subset markers
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Parallel comparison of SOCE kinetics and amplitude
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Subset-specific gene expression analysis of ORAI1 and regulators
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Functional outcomes assessment (proliferation, cytokine production)
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In vivo subset tracking after adoptive transfer
These approaches can reveal how ORAI1 contributes to the specialized functions of different T cell populations in chicken immune responses .
